Original Article LIN28B Suppresses Microrna Let-7B Expression to Promote CD44+/LIN28B+ Human Pancreatic Cancer Stem Cell Proliferation and Invasion
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Am J Cancer Res 2015;5(9):2643-2659 www.ajcr.us /ISSN:2156-6976/ajcr0008500 Original Article LIN28B suppresses microRNA let-7b expression to promote CD44+/LIN28B+ human pancreatic cancer stem cell proliferation and invasion Yebo Shao1*, Lei Zhang1*, Lei Cui2*, Wenhui Lou1, Dansong Wang1, Weiqi Lu1, Dayong Jin1, Te Liu3 1Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, China; 2Department of General Surgery, Jiangsu University Affiliated Hospital, Zhengjiang 212000, China; 3Shanghai Geriatric Institute of Chinese Medicine, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200031, China. *Equal contributors. Received March 27, 2015; Accepted July 5, 2015; Epub August 15, 2015; Published September 1, 2015 Abstract: Although the highly proliferative, migratory, and multi-drug resistant phenotype of human pancreatic can- cer stem cells (PCSCs) is well characterized, knowledge of their biological mechanisms is limited. We used CD44 and LIN28B as markers to screen, isolate, and enrich CSCs from human primary pancreatic cancer. Using flow cytometry, we identified a human primary pancreatic cancer cell (PCC) subpopulation expressing high levels of both CD44 and LIN28B. CD44+/LIN28B+ PCSCs expressed high levels of stemness marker genes and possessed higher migratory and invasive ability than CD44-/LIN28B- PCCs. CD44+/LIN28B+ PCSCs were more resistant to growth inhibition induced by the chemotherapeutic drugs cisplatin and gemcitabine hydrochloride, and readily established tumors in vivo in a relatively short time. Moreover, microarray analysis revealed significant differences between the cDNA expression patterns of CD44+/LIN28B+ PCSCs and CD44-/LIN28B- PCCs. Following siRNA interference of endogenous LIN28B gene expression in CD44+/LIN28B+ PCSCs, not only was their proliferation decreased, there was also cell cycle arrest due to suppression of cyclin D1 expression following the stimulation of miRNA let-7b expression. In conclusion, CD44+/LIN28B+ cells, which possess CSC characteristics, can be reliably sorted from human primary PCCs and represent a valuable model for studying cancer cell physiology and multi-drug resistance. Keywords: Pancreatic ductal adenocarcinoma, cancer stem cells, CD44, Lin28B Introduction properties; furthermore, the capacity for tumor formation and growth resides exclusively in a Pancreatic ductal adenocarcinoma (PDAC) is a small proportion of tumor cells, termed cancer highly lethal malignancy that is usually diag- stem cells (CSCs) [7-11]. Pancreatic CSCs nosed at a late stage, at which optimal thera- (PCSCs) were first characterized by Li et al. 6[ ] peutic options have been skipped [1]. It is one and were shown to be not only highly tumori- of the most chemoresistant tumors; the surviv- genic, but also possessed the ability to self- al rate is < 5% [2]. PDAC is not only notorious renew and produce differentiated progeny that for being difficult to diagnose at an early stage reflected the heterogeneity of the patient’s pri- and its poor recurrence-free prognosis, but mary tumor [3]. Moreover, an increasing num- also the lack of effective treatment thereof and ber of studies have reported human pancreatic limited knowledge of its biological characteris- cancer cell subpopulations, such as CD31+/ tics [3, 4]. Thus, there is an urgent need for bet- CD45+ [12]; Hoechst 33342-/CD133+/ALDH1+ ter understanding of the cellular/molecular [1]; ESA+/CD44+ [3]; and CD24+/CD44+ [4], properties associated with PDAC to explore which express CSC-associated characteristics novel venues of diagnosis and treatment of this and stem cell markers [5]. There are several disease [1, 5, 6]. Recent evidence suggests prominent CSC characteristics: they (a) self- that tumors consist of heterogeneous cell pop- renew and are highly clonogenic, (b) differenti- ulations, which possess different biological ate in vitro to form organized spheroids in sus- LIN28B promotion of PCSC proliferation and invasion pension, (c) express multipotency and tissue- We demonstrated a CD44+/LIN28B+ PCSC specific differentiation markers, (d) generate subpopulation that proliferates rapidly and tumors in vivo through self-renewal mecha- exhibits multi-drug resistance, high invasion nisms, (e) undergo in vivo differentiation to pro- ability, and adherin. Therefore, CD44+/LIN28B+ duce a disease similar to that in the patient PCSCs represent a potentially powerful in vitro [13]. The observation that stem cells and some model for studying cancer cell metastasis, inva- CSCs share the common defining features of sion, and self-renewal and for assessing the incompletely differentiated state and self- effectiveness of novel therapeutics for PDAC. renewal capacity led to the CSC hypothesis as a possible mechanism for total tumor growth Materials and methods as the result of the proliferation of a small sub- Isolation CD44 and LIN28B phenotype cells by population of cells [9-11, 14]. magnetic activated cell sorting system Lin28, which is an RNA-binding protein, regu- lates cell growth and differentiation [15]. CD44+ and LIN28B+ subpopulation cells were Developmental timing in Caenorhabditis ele- isolated from primary cancer cells from pancre- gans is regulated by a heterochronic gene path- atic cancer tissues using 4 μl of the primary monoclonal antibodies (rabbit anti-human way. The heterochronic gene liN28 is a key LIN28B-FITC, rabbit anti-human CD44-PE, regulator early in the pathway [16]. liN28 eBioscience) stored at 4°C in PBS for 30 min in encodes an approximately 25-kDa protein with a volume of 1 ml as previously described [7, two RNA-binding motifs: a so-called “cold shock 21]. After reaction, the cells were washed twice domain” (CSD) and a pair of retroviral-type in PBS, and were put the secondary monoclo- CCHC zinc fingers; it is the only known animal nal antibodies (Goat anti-rabbit coupled to protein with this motif pairing. The CSD is a magnetic microbeads, Miltenyi Biotec, Auburn, β-barrel structure that binds single-stranded CA), incubated at 10°C in PBS for 15 min and nucleic acids [16]. Lin28 inhibits the biogene- then washed twice in PBS. Single cells were sis of a group of microRNAs (miRNAs), among plated at 1000 cells/ml in DMEM: F12 which are the let-7 family miRNAs shown to par- (HyClone), supplemented with 10 ng/ml basic ticipate in regulation of the expression of genes fibroblast growth factor (bFGF), 10 ng/ml epi- involved in cell growth and differentiation [17]. dermal growth factor (EGF), 5 μg/ml insulin and The mechanism underlying selective let-7 inhi- 0.5% bovine serum albumin (BSA) (all from bition by Lin28 has been studied extensively. Sigma-Aldrich). All CD44+/LIN28B+ cells were The common theme is that Lin28 binds to the cultured in above conditions as non-adherent terminal loop region of pri/pre-let-7 and blocks spherical clusters which were called PCSCs, their processing [15]. The miRNAs are small and CD44-/LIN28B- cells which were cultured RNA molecules (21-23 nucleotides) that act as under general conditions as adherent clusters, negative regulators of gene expression either was called PCCs. All Cells had been cultured on by blocking mRNA translation into protein or the same conditions until passage 4th before through RNA interference [18-21]. Previous making ulterior experiments. The methods studies have reported that dysregulation of were carried out in accordance with the specific miRNAs is associated with certain approved guidelines. types of cancer, and they are thought to act as either oncogenes or tumor suppressors Quantitative Real-time PCR (qRT-PCR) analysis depending on the target gene [19, 21, 22]. Furthermore, the miRNA let-7b regulates self- Total RNA from each cells was isolated using renewal of embryonic stem cells and the prolif- Trizol Reagent (Invitrogen) according to the eration and tumorigenicity of cancer cells by manufacturer’s protocol. The RNA samples inhibiting cyclin D1 (CCND1) expression were treated with Dnase I (Sigma-Aldrich), [23-25]. quantified, and reverse-transcribed into cDNA using the ReverTra Ace-α First Strand cDNA In view of the above findings, we sorted a novel Synthesis Kit (TOYOBO). qRT-PCR was conduct- CSC subpopulation overexpressing CD44 and ed using a RealPlex4 real-time PCR detection LIN28B at the cell surface (CD44+/LIN28B+) system from Eppendorf Co. LTD (Germany), from human primary pancreatic cancer tissues. with SyBR Green RealTime PCR Master MIX 2644 Am J Cancer Res 2015;5(9):2643-2659 LIN28B promotion of PCSC proliferation and invasion (TOYOBO) used as the detection dye. qRT-PCR DAPI (Sigma-Aldrich) at room temperature for amplification was performed over 40 cycles 30 min. Then the cells were thoroughly washed with denaturation at 95°C for 15 sec and with TBST and viewed through a fluorescence annealing at 58°C for 45 sec. Target cDNA was microscope (DMI3000; Leica, Allendale, NJ, quantified using the relative quantification USA). method. A comparative threshold cycle (Ct) was used to determine gene expression relative to a Soft agar colony formation assay control (calibrator) and steady-state mRNA lev- els are reported as an n-fold difference relative All steps were according to the previously to the calibrator. For each sample, the maker described [19]. Soft Agar Assays were con- genes Ct values were normalized using the for- structed in 6-well plates. The base layer of each mula ΔCt = Ct_markers-Ct_18sRNA. To deter- well consisted of 2 mL with final concentrations mine relative expression levels, the following of 1 × media (DMEM+10% FBS) and 0.6% low formula was used ΔΔCt = ΔCt_CSCs-ΔCt_CCs. melting point agarose. Plates were chilled at The values used to plot relative expressions of 4°C until solid. Upon this, a 1.0 ml growth agar 4 markers were calculated using the expression layer was poured, consisting of 1 × 10 cells 2-ΔΔCt. The mRNA levels were calibrated based suspended in 1 × media and 0.3% low melting on levels of 18 s RNA. The cDNA of each stem point agarose. Plates were again chilled at 4°C cell markers was amplified using primers as until the growth layer congealed.